Biochimica et Biophysica Acta, 446 (1976) 1-9 © Elsevier/North-Holland Biomedical Press

BBA 37448 T H E A M I N O A C I D S E Q U E N C E OF T H R E E N O N - C U R A R I M I M E T I C T O X I N S F R O M NAJA NIVEA V E N O M *

DAWIE P. BOTES and CORNELIS C. VILJOEN National Chemical Research Laboratory, Councilfor Scientific and Industrial Research,P.O. Box 395, Pretoria, 0001 (Republic of South Africa) (Received April 9th, 1976)

SUMMARY Three toxins of the non-curarimimetic type have been isolated from the venom of the Cape cobra Naja nivea. 3~he basic and hydrophobic amino acids are dominant in all three toxins. They comprise 60 amino acid residues with 4 intrachain disulphide linkages. The toxins have been characterized with respect to their linear structures and immunochemical properties. Toxicity and hemolytic data suggest a much higher affinity for receptors on the heart cell membrane than for that of the red cell.

INTRODUCTION The so-called "membrane-active" polypeptides, the presence of which is not unambiguously established in Dendroaspis venoms, constitute a rather abundant proportion of Naja and Haemachatus venoms [1 ]. Thus, in Naja mossarnbica mossambica venom, 69 %o of the proteinaceous material of the venom is accounted for by "membrane-active" polypeptides [2]. The intravenous LDs0-values of these polypeptides are an order of magnitude higher than the corresponding values for the more potent neurotoxins [3]. Subcutaneously, due to adsorption at the site of the injection, the toxicity of these former compounds are lower by another order of magnitude [4]. Their negligible moment in natural occurring snake envenomation, supplemented by the quenching effect of the neurotoxicity of Elapid venoms, contribute to the facet that the " m e m b r a n e active" polypeptides were often overlooked. Nevertheless, Epstein [5], as early as 1930, recognized the early death in the cat, caused by a large dose of Naja nivea venom, to be due to cardiac failure. Christensen [6] isolated from the latter venom, in addition to the two major neurotoxins which he called the a and ~ toxins, a third toxic component, designated toxin ~, which, by virtue of the tenfold difference in intravenous and subcutaneous LDs0-values, probably constitute a cardiotoxic fraction. More recently a chromatographic proce* Supplementary data to this article, giving details of the peptide sequences of toxins Vnl, VU2 and V"3, are deposited with, and can be obtained from: Elsevier Scientific Publishing Company, BBA Data Deposition, P.O. Box 1527, Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/046/37448/446 (1976) 1.

dure for tile isolation of toxins a and/3 and a hitherto unknown toxin 6 has been described [7]. The linear structure of toxin 6 [7] and subsequently also those of toxins a and/3 and the positioning of the disulphide bridges of toxin a, have been reported [8]. The present study extends the structural investigations of Naja nivea venom components to the major "membrane-active" polypeptides of this venom. EXPERIMENTAL PROCEDURE Lyophilized Naja nivea venom was obtained from D. Muller, Professional Snake Catcher (Pty) Ltd., 215 Barkston Drive, Blairgowrie, Johannesburg, 2001, Republic of South Africa. 1,4-Dithiothreitol and trifluoracetic acid were products of Merck AG (Germany), while phenylisothiocyanate was bought from Fluka AG (Switzerland). 5,5-Dithiobis-(2-nitrobenzoic acid) was ordered from K & K Laboratories (New York, U.S.A.). Trypsin was supplied by Seravac Laboratories (Cape Town, Republic of South Africa) as a twice crystallized diphenyl carbamyl chloride-treated, salt-free preparation, a-Chymotrypsin (3 × crystallized) was purchased from Worthington. DEAE-cellulose DE-52 and CM-cellulose CM-52 were obtained from Whatman (England) while Sephadex was a product of Pharmacia. The Sephadex and ionexchange celluloses for column chromatography, were prepared as recommended by the manufacturer. Reduction of the toxins with dithiothreitol, S-carboxymethylation with iodoacetic acid and amino acid analyses, were performed as described previously [9]. Tryptophan was determined spectrophotometrically on toxin samples by the method of Goodwin and Morton [10]. Determination of free sulfhydryl groups was carried out in the absence and presence of 6 M guanidine hydrochloride according to the description by Ellman [11]. Reduced and S-carboxymethylated toxin was digested with chymotrypsin or trypsin, at 37 °C for 2 h in 2 ~ NH4HCO3 and an enzyme to substrate ratio of 1:100 (w/w). Digests were chromatographed on DEAE-cellulose as a first separation step and the peptides further purified where applicable, by high voltage electrophoresis or paper chromatography according to a procedure reported previously [12]. Sequence analyses on the NH2-terminal segments of the toxins were carried out by the automated Edman Technique while peptides were sequenced by a manual manipulation of the Edman degradation procedure as described in a previous paper [12]. Phenylthiohydantoin derivatives of amino acids were identified by thin layer chromatography and gas chromatography [12]. lmmunochemical properties of the toxins were examined by gel diffusion and toxicity assays on the different toxins carried out as described [13]. Hemolytic activity of the toxins were followed by a modification (Visser, L. and Louw, A. J., unpublished) of a method employed for complement lysis [14]. RESULTS Fig. 1 depicts the elution profile obtained for the separation of 10 g of crude venom on Sephadex G-50. Fraction III contained inter alia the toxins, which are the subject of the present report. The toxic principles were separated on CM-cellulose using a linear ammonium bicarbonate gradient (Fig. 2). Toxin VU3 separated in pure

1.25

I ,OC

Q75

0.5C

0.25

I 2000

I 2500

I 3000

I 3500

I 4000

I 4500

I 5000

ELUATE VOLUME(too

Fig. 1. Elution profile for the separation of 10 g of crude venom on Sephadex G-50 (450 × 3.8 cm) in 0.1 M ammonium bicarbonate solution.

lira

oi

V~3

04

02

9

•'o

,~

.'o

~

.~

%GRt¢IENT

Fig. 2. Chromatography of fraction III (Fig. 1) on CM-cellulose (50 × 3.8 cm). Lyophilized fraction III (9 g) was loaded on the column and elution effected by a linear gradient of 0.01 M-0.4 M ammonium bicarbonate solution over 16 1.

f o r m in this step. Toxins V " l a n d V " 2 c o u l d be purified by further f r a c t i o n a t i o n o f p e a k V (Fig. 2) on CM-cellulose at p H 4.5 (Fig. 3). Toxins VUl, V " 2 a n d V " 3 app e a r e d to be h o m o g e n e o u s b y a m i n o acid analysis, a m i n o - t e r m i n a l end g r o u p determ i n a t i o n a n d i m m u n o d i f f u s i o n . E x a m i n a t i o n o f the toxins with E l l m a n ' s reagent b o t h in the presence a n d absence o f d e n a t u r i n g agent, showed the fractions to be devoid o f free sulfhydryl groups.

0"5 I

VII2

]3

0.4

g

~

V

t

0.3-

0.2-

0.t

I 40

30

I 50

I 60

70

%GRADIENT F i g . 3. F r a c t i o n a t i o n

o f f r a c t i o n V ( F i g . 2) o n C M - c e ] l u ] o s e ( 5 0 × ] . 9 c m ) . L y o p h i l i z e d

fraction V

(1 g) was dissolved in starting buffer and applied to the column. Gradient elution from 0.1 M-0.35 M ammonium acetate pH 4.5 over 8 1.

Alignment of Peptides Toxin V111(Fig. 4). Sequencing the NH2-terminal segment of the reduced and S-carboxymethylated toxin by means of the sequencer, provided directly the positions of peptides T-1 to T-7 and established the overlap between peptides T-6 (a), T-7 and T-8. Since peptide T-10 was the only peptide not terminating in either lysine or arginine, this peptide was assigned as the carboxyl terminal peptide of toxin V"I. Peptide T-9 could then be positioned by difference, amino-terminal to peptide T-10. Toxin V"2 (Fig. 5). The sequence of the amino-terminal segment of toxin V"2 allowed peptides T-1 to T-7 (a) to be placed in that order from the NH2-terminus. Peptide T-9 ,'ontained no terminal argine or lysine and must therefore be derived from

qO

20

NH2- Leu -Lys - Cys - His - Lys - Leu -Vol - Pro - Pro - VoI-Trp - Lys -Th r-Cys-Pro- Glu-G ly-Lys - Ash - Leu o-T~ ~ T 2 =~ ,=' T3 =. ~ T4 ---~T5 = SEQUENCER

30

40

Cys - T y r - Lys-Met-Phe-Met-VoI-Ser-Thr-Ser-Thr- VaI-Pro- V o I - L y s - A r g - G l y - C y s - I i e - A i p ~T5 ~ ,= T6 b -T8

q

SEQUENCER 50

T6 (a)

"=T~,'~" q

m

T8 (o) 60

V o l - Cys-Pro- Lys-A~o-Ser-AIo - Leu -Vo I- Lyl-'~/r -Vol - Cys - Cys -Ser -Thr -Asp-Lys- Cys-Asn T8 • I T9 --TtO =.. T8(o )..---=-

Fig. 4. Amino acid sequence of toxin V"I.

I0

20

NH2-Leu- Ly~-Cys- H i s - G i n - Leu - ~.le - P r o - Pro- P h e - T r p - L y s - T h r - C y l - P r o - G l u - GI y - L y s - A s n - Leu SEQUENCER

30

40

C y s - T y r - L y s - Met - T y r - M e t - V o l - A I o - T h r - P r o - M I t - T I e - P r o - V a I - L y s - A r g - G l y - C y s - T l e - A s p ~ T 4 ~ =1 T5 91DT~_ i i T7(O) T7 SEQUENCER

It

50

60

Vol - C y s - P r o - l_ys- A s n - S e r - A l o - L e u - V o l - L y s - T y r - M e t - C y s - C y s - A s n - T h r - Asp - Lys- Cys-Asn ~T7

( o .~ T7

~ -~

T8

~

T9

I~

Fig. 5. The amino acid sequence of toxin V'2.

the COOH-terminus of toxin VII2. Peptide T-8 was positioned by difference, NH2terminal to peptide T-9. Although no overlap was established between peptides T-9 and T-10 in the sequence of VH1 or between peptides T-8 and T-9 in VII2, the possibility of missing peptides in this region was excluded since in both instances, the complete amino acid composition could be accounted for by the tryptic peptides. Toxin P"3 (Fig. 6). By amino acid composition, tryptic peptides T-1 to T-5 and chymotryptic peptides C-1 to C-3 could be positioned from the aminoterminal sequence of toxin VII3. The amino-terminal sequence also provided the necessary overlap between peptides T-5 and T-6. Peptide T-9 was assigned COOH-terminal Io

20

~ll-12-Leu-Lys-Cys-Asn-GIn - Leu - ' r l e - Pro- P r o - P h e - T r p - L y s - T h r - C y s - P r o - L y s - G l y - L y s - A s n - L e a ,,,s--T( ~ c II ~_ c t (o).~.=.. =

T2 cI C~(b)

4

v

c2 C2(a)

--

I=

SEQUENCER

50

40

Cys - Tyr - A s n - Met-'ryr- Met-VoI.Ser-Thr -Ser - T h r - V a l - Pro-VaI-Lys-Arg- Gly- Cys-Ile- AspT5 ~- ~ T6(o) - - -T7 T6 L ~C2 ~ C5,--~ C4 q"C2(b~'

~ SEQUENCER

C4 (o) == 50

60

Vol - Cys-Pro - Lys - A=n - S e t - A I o - Leu - Vo I - Lys - T y r - Vo I- Cys - Cys - A s n - T h r - A s p - A r g - C y s - A s n T7 ~ T8 ~ ~ T9 I¢ C4 =- ~ CS =-

C4(a )

.~- ~

C4 (b),-D,

Fig. 6. Amino acid sequence of toxin V"3.

TABLE I TOXICITY AND HEMOLYTIC ACTIVITIES OF TOXINS Vlq, Vtl2 AND VII3 Toxin

Toxicity 5 ~ Fiducial Hemolytic activity* (t,g/g mouse) limits

WUl VH2

2.9 1.5 1.6

VtI3

2.6-3.1 1.4-1.7 1.4-1.8

1.6 1.6 1.4

* Expressed as a ~ of the hemolytic activity ofNaja mossambicamossambicaV~2 under identical assay conditions. since it was the only tryptic peptide not terminating in lysine or arginine. The positioning of peptide T-8 amino-terminal to peptide T-9, was by virtue of the overlaps provided by peptides C-4 (b) and C-5. That left the remaining peptide, T-7, to be positioned by difference, amino-terminal to peptide T-8. DISCUSSION Designations such as direct lytic factor [15], cobramine A and B [16], toxin 7 [17], cardiotoxin [18], cytotoxin I [19] and II [20], skeletal muscle depolarizing factor [21], etc., document the earlier held contention that the observed functionally divergent properties were all associated with structurally different venom components. Suggestions as to their possible identity and collective grouping were advanced by two different schools [22, 23]. Recognizing the apparent common feature shared by these compounds, i.e., their action at the level of the cell membrane, leading to disturbance of its organization and function, Condrea [24] suggested the unifying term "membrane-active polypeptides" to denote this particular group of venom components. Such a denomination however, suggests a rather unspecific mode of action and ignores a possible selective affinity for receptors on different types of membranes such as erythrocytes, heart cells etc. The three Naja nivea components, the subject of this study, for instance, exhibit very weak hemolytic activity even when tested on the most sensitive red cells, those of the guinea pig. In fact, such a high concentration of these components are required to produce a measurable response that the effect of a small amount of a lytic impurity, if present, could become significant. The membrane activity of the Naja nivea toxins, as manifested by hemolysis, are two orders of magnitude lower than that of Naja mossambica mossambica VU2. Their LDso-values however, are on par with other members in the group. Evidence of their cardiotoxic nature is the simultaneous arrest of both respiration and cardiac activity produced by these substances in the cat, in contrast to the prolonged cardiac output after cessation of respiration produced by toxin fl, a typical neurotoxin from the same venom (Kundig, H. and Botes, D. P., unpublished). Whether heart cell membrane processes are involved in the observed compromised cardiac output and eventual arrest, remains to be investigated for the three Naja nivea components. It is however evident that the heart cell membrane, if it is the membrane function that is involved, is either far more sensitive to these latter compounds than the guinea pig red cell, or hemolysis is no measure of the specific interaction of these group of compounds with the red cell membrane and certainly not of their physiologically significant action.

A bifunctional nature inherent in the structure of some members of this group of venom components would explain the variance in hemolysis patterns observed. The structural feature common to all members of this group would allow recognition of specific receptors on the heart cell membrane. The second feature, either detergentlike or more specific in its recognition of a receptor on the red cell membrane, responsible for disruption of the membrane or activation of endogeneous membranebound substances, giving rise to phenomena such as hemolysis, is expressed in some members of the group only. The three Naja nivea toxins, the linear structures of which are depicted in Figs. 4-6, are the most abundant of the non-curariform toxins in this venom. They account for 2 0 ~ , 8.4~o and 1.2~o of the whole venom and are designated VH1, V~2 and V~3 respectively, in accordance with a recent proposal [25]. The typical membrane-active features, with the preponderance of basic and hydrophobic residues and the replacement of the Cys-Tyr-X-Lys-X-Trp sequence, characteristic of the venom neurotoxins, by Cys-Tyr-Lys-Met-X-Met in the region 21-26, and the lack of the Arg-Gly sequence, common to all curarimetic neurotoxins, are present in the Naja nivea toxins. In accordance with Boquet's [26] classification, the Naja nivea toxins are immunochemically distinct from both types of neurotoxins (Fig. 7). Amongst themselves,

Fig. 7. Comparison of toxins VtI1, V~2 and V"3 with Naja nivea toxins a and fl and Naja mossambica mossambica toxin VH1 by immunodiffusion. The central wells contained polyvalent antivenin and the other wells the toxins as indicated. 1, 2 and 3 signify Naja nivea toxins Vlq, V~2 and VH3 respectively, a and fl the corresponding neurotoxins from the same venom and C, toxin VItl from Naja rnossambica mossambica.

VH1 shows weak spurring against V1X2 and identity to VII3. Similarly, VH2 and VH3 are indistinguishable from each other. Against Naja mossambica mossambica Vii1, Naja nivea VIq, shows strong spurring, VH2 weaker spurring and Vii3 exhibits identity to the Naja mossambica mossambica toxin. The apparent identity of Na./a nivea VH3 to both VH1 from the same venom and the Naja mossambica mossambica toxin, considering the strong spurring between the latter two toxins, is not immediately apparent. A possible reason is the failure of the non-common antigenic determinants to form a precipitin band either through their positional distribution or the subcritical abundance of these determinants to allow lattice formation with the homologous antibodies. The functional difference of these toxins in their ability to lyse red ceils is obviously not expressed as a structurally distinguishable antigenic determinant. The linear sequences of 35 membrane active-type polypeptides are presently known [18-20, 27-39]. All of these have been isolated from Haemachatus and Naja venoms. The presence of a cardiotoxin in Dendroaspis jamesoni venom has recently been suggested [40]. However, by immunodiffusion, the presence of an antigen similar to Naja nuvea toxin V II 1 could not be demonstrated in Dendroaspis jamesoni venom (Strydom, A. J. C. and Botes, D. P., unpublished). If therefore, components similar in pharmacological action to the Haemachatus and Naja cardiotoxins are present in Dentroaspis jamesoni venom, their structural features must be considerably different from their counterparts in Naja and Haemachatus venoms. However, in most venoms the neurotoxins exist in two immunochemically distinct forms. The existence of a second hitherto unknown membrane-active type toxin is therefore not excluded. The structural basis for the observed disparity of different toxins in their ability to lyse red ceils is by no means obvious from a comparison of the known structures. However, certain structural features which could impart an affinity for nonpolar regions on membranes are emerging from such a comparison. The concerved hydrophobicity in the region 6-10 and largely also in positions 22-27, 32-34 and 47-49 possibly suggests the existence of a non-polar pocket or patch in the three-dimensional structure which could interact with the cell-membrane. An interaction of this type is exemplified by the binding of insulin with the receptor on the membrane surface [41 ]. Further strict structural requirements seem to be the invariant sequences Asn-LeuCys-Tyr(Phe), Arg-Gly-Cys and Cys-Pro-Lys in positions 13-15, 19-22, 36-38 and 42-44 respectively. Noteworthy is the fact that in all the latter instances a half-cystine residue is involved, implying the necessity for conformational restraints around these half-cystine residues, not only for Correct chain folding but also for expression of activity on membranes. High resolution X-ray diffraction seems the only available tool at the present to reveal subtle structural differences responsible for the observed functional divergence amongst members of this group of venom components. ACKNOWLEDGEM ENTS We wish to acknowledge the very expert assistance of Mrs Helen Kruger with all aspects of this project. We are greatly indebted to Drs. L. Visser and A. I. Louw for the hemolytic assays and many fruitful discussions. We also wish to express our sincere appreciation to Dr P. A. Christensen of the SAIMR for a generous gift of polyvalent antivenin.

REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Lee, C. Y. (1972) Annu. Rev. Pharmacol. 12, 265-286 Louw, A. I. (1974) Biochem. Biophys. Acta 336, 470-480 Lee, C. Y. (1972) Annu. Rev. Pharmacol. 12, 84-105 Tseng, L. F., Chiu, T. H. and Lee, C. Y. (1968) Toxicol. Appl. Pharmacol. 12:526-536 Epstein, D. (1930) Q. J. Exper. Physiol. 20, 7-19 Christensen, P. A. (1953) Bull. World Hlth. Org. 9, 353-370 Bores, D. P., Strydom, D. J., Anderson, C. G. and Christensen, P. A. (1971) J. Biol. Chem. 246, 3132-3139 Botes, D. P. (1971) J. Biol. Chem. 246, 7383-7391 Botes, D. P. and Viljoen, C. C. (1974) Toxieon 12, 611-619 Goodwin, T. W. and Morton, R. A. (1946) Biochem. J. 40, 628-632 Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 Botes, D. P. and Viljoen, C. C. (1974) J. Biol. Chem. 249, 3827-3835 Botes, D. P. (1974) Biochim. Biophys. Acta 359, 242-247 Boackle, R. J. and Pruitt, K. M. (1974) in Proceedings of the XXI Colloquim: Protides of the Biological Fluids (Peeters, H., ed.), pp. 613-617, Pergamon Press, Oxford Aloof-Hirsch, S., De Vries, A. and Berger, A. (1968) Biochim. Biophys. Acta 154, 53-60 Reed-Larsen, P. and Wolff, J. (1968) J. Biol. Chem. 243, 1283-1289 lzard, Y., Boquet, M., Ronsseray, A. and Boquet, P. (1969) C.R. Acad. Sci., Ser. D. 269, 96-97 Narita, K. and Lee, C. Y. (1970) Biochem. Biophys. Res. Commun. 41,339-343 Hayashi, K., Takechi, M. and Sasaki, T. (1971)Biochem. Biophys. Res. Commun. 45, 1357-1362 Takechi, M. and Hayashi, K. and Sasaki, T. (1972) Mol. Pnarmacol. 8, 446--451 Meldrum, B. S. (1963) J. Physiol. Lond. 168, 49p Slotta, K. H. and Vick, J. A. (1969) Toxicon 6, 167-173 Lee, C. Y., Lin, J. S. and Wei, J. W. (1971) in Toxins of Animal and Plant Origin (De Vries, A. and Kochva, E., eds.), Vol. 1, pp. 307-318, Gordon and Breach, New York, London and Paris Condrea, E. (1974) Experientia 30, 121-129 Botes, D. P. Carlsson, F. H. H., Joubert, F. J., Louw, A. I., Strydom, A. J. C., Strydom, D. J. and Viljoen, C. C. (1974) Toxicon 12, 99-101 Boquet, P., lzard, Y. and Ronsseray, A. M. (1970) C.R. Acad. Sci. 271, 1456-1459 Carlsson, F. H. H. and Joubert, F. J. (1974) Biochim. Biophys. Acta 336, 453-469 Grishin, E. V., Sukhikh, A. P., Adamovich, T. B., Ovchinnikov, Yu. A. and Yukelson, L. Ya. (1974) FEBS Lett. 48, 179-183 Louw, A. I. (1974) Biochim. Biophys. Acta 336, 481495 Louw, A. I. (1974) Biochem. Biophys. Res. Commun. 58, 1022-1029 Weise, K. H. K., Carlsson, F. H. H., Joubert, F. J. and Strydom, D. J. (1973) Hoppe Seyler)s Z. Physiol. Chem. 354, 1317-1328 Frykland, L. and Eaker, D. (1973) Biochemistry 12, 661-667 Carlsson, F. H. H. (1974) Biochem. Biophys. Res. Commun. 59, 269-276 Frykland, L. and Eaker, D. (1975) Biochemistry 14, 2860-2865 Frykland, L. and Eaker, D. (1975) Biochemistry 14, 2865-2871 Shipolini, R. A. Kissonerghis, M. and Banks, B. E. C. (1975) Eur. J. Biochem. 56, 449454 Joubert, F. J. (1975) Hoppe Seyler~s Z. Physiol. Chem. 356, 1893-1900 Joubert, F. J. (1975) Hoppe Seyler~s Z. Physiol. Chem. 356, 1901-1908 Joubert, F. J. (1976) Eur. J. Biochem. in the Press Patel, R. and Excell, B. J. (1974) Toxicon 12, 577-585 Pullen, R. A., Lindsay, D. G., Wood, S. P., Tickle, I. J., Blundell, T. L., Wollmer, A., Krail, G., Brandenburg, D., Zahn, H., Gliemann, J. and Gammeltoft, S. (1975) Nature 259, 369-373